A central challenge in neurobiology is the controlled activation or silencing of defined neural circuits in the living animal to investigate the functions of these circuits. A number of technologies have been devised to this end, including optogenetic and chemical genetic approaches. Among the latter we have developed a synthetic signaling system activated by an inert drug, which we named DREADD (Designer Receptors Exclusively Activated by a Designer Drug). DREADDs are mutant G protein coupled receptors which lost the ability to respond to their natural ligand and which respond to a pharmacologically inert, drug-like compound (CNO) with nanomolar potency. Currently, the existing DREADDs cover the Gi, Gq, and Gs signaling pathways. These modified GPCRs can be utilized as ideal tools for remotely controlling the activity of discrete populations of neurons in vivo. We have demonstrated the power of this approach in several proof-of-concept studies. In order to fully explore the in vivo applications of the DREADD technology we propose here to generate a widely applicable transgenic platform by creating hM3Dq, rM3Ds, as well as the G-coupled hM4Di strains of mice which allow for the Cre recombinase-mediated restricted expression of these pathway-selective DREADDs. Specifically, we will generate C57BL/6 mouse lines carrying within the ROSA26 locus a ubiquitously expressed promoter allowing Cre-mediated expression of Dq and Ds, as well as Di, upon removal of a floxed STOP (Aim 1). We will then characterize these lines after crossing to a Cre driver inducing neuron-specific expression of Dq and Ds, as well as Di, after Cre-mediated excision of the floxed STOP (Aim 2). Our analyses will insure that each line has robust expression in the presence and no background expression of the respective DREADD in the absence of Cre, and that the Cre-induced expression of each DREADD is consistent with the pattern of expression of the Cre driver. We will characterize the functionality of the DREADD lines in vitro to insure that Cre-mediated expression of each DREADD leads to neuronal activation or silencing upon CNO application;that activation or silencing occurs only in the presence of CNO, but not in its absence;and only in cells expressing Cre and not in control cells. Finally, we will test functionality in vivo by assessing behavioral effects of DREADD:Cre mice with increasing doses of peripherally administered CNO. The deliverables at the end of our project will be three fully characterized DREADD mouse lines ready for mating with any Cre driver line. With the ever growing number of Cre driver lines becoming available these DREADD lines will be applicable to studying a wide array of research questions. Our project will thus advance neuronal circuit analysis efforts by establishing a generic platform for the highly specific and remote control of neuronal activity and signaling in transgenic mice. PUBLIC HEALTH RELEVANCE: Abnormal neural circuitry is likely at the core of many neuropsychiatric diseases. The goal of this proposal is to advance the methods of analysis, and the resulting increased understanding, of how neural circuits in the mammalian brain work. Thus this proposal for developing a widely applicable transgenic platform for the analysis of neural circuitry targets areas of fundamental importance to public health.